Angiogenesis, or the development of new blood vessels from pre-existing blood vessels, is an essential feature of tissue development and wound healing. Without the appropriate development of a blood supply, tissues cannot survive. The circulatory system is essential for the supply of oxygen and nutrients to tissues and for the removal of by-products of metabolism.
Angiogenesis is normally observed in wound healing, fetal and embryonal development, and formation of the corpus luteum, endometrium and placenta. The control of angiogenesis is a highly regulated system of angiogenic stimulators and inhibitors. Thus, angiogenesis is a critical component of the body's normal physiology. In adults, angiogenesis is a relatively rare occurrence except during wound healing and in the ovaries during the sexual cycle.
However, there are a number of “angiogenesis-dependent diseases” in adults where angiogenesis is important. The most important of these is the angiogenesis associated with the growth of solid tumors, hemaniomas, proliferative retinopathies and rheumatoid arthritis. Certain disease states can alter the control of angiogenesis and, in many cases, the pathological damage associated with the disease is related to uncontrolled angiogenesis. Uncontrolled angiogenesis can act detrimentally when blood vessels multiply and enhance the growth and metastasis of tumors. Aberrant angiogenesis is also associated with numerous disorders, including rheumatoid arthritis, where blood vessels invade the joint and destroy cartilage, and numerous ophthalmologic pathologies, such as diabetic retinopathies in which new capillaries invade the vitreous, bleed and cause blindness. Angiogenesis can also play a significant part in other diseases, such as coronary artery disease and restenosis following angioplasty.
Angiogenesis is essential to tumor development and growth. An angiogenesis inhibitor may therefore stop or inhibit the growth of primary tumors, impede or reduce the formation of metastases, and impede the appearance of secondary growths. Angiogenic inhibitors are also useful in the treatment of non-neoplastic disorders in which an angiogenic activity occurs.
The development of angiogenesis inhibitors can provide a means for controlling these diseases. Compounds that have anti-angiogenic activity can be used, for example, as anti-tumor agents and for the treatment of ophthalmic disorders, particularly involving the retina and vitreous humor, and for hyperproliferative dermatological disorders, such as psoriasis, that have an angiogenic component. Thus, there is an important need to identify compounds that enhance angiogenesis, and compounds that inhibit angiogenesis.
Assays and methods to study angiogenesis can be conducted either in vivo or in vitro. An in vitro method can include the method described in U.S. Pat. No. 5,976,782, issued to Parish at al. on Nov. 2, 1999, related to determining angiogenesis by culturing a blood vessel fragment in a physiological gel, and testing substances on the gel to determine their angiogenic effects. Other in vitro assays have usually entailed establishing long term cultures of endothelial cells and inducing formation of microvessels by placing the cells on extracellular matrices or exposing the cells to various angiogenic stimuli. Such assays are highly artificial and may not represent a physiological response, particularly as the endothelial cells are already activated, having been cultured for considerable periods of time in the presence of growth factors before use.
There remain significant drawbacks to in vitro assays and methods. An in vitro sample of a tissue is disconnected from the remainder of the body system, prohibiting stimulation to, or stimulation from, other body tissues, compounds, and functions. The in vitro method provides no signal of the cellular stimuli caused by the test compound to the body, and no feedback from the body to the affected cells. Therefore, while there are unique advantages to the use of an in vitro assay, in vivo studies remain as an important assay in the development of medical drugs and products.
Three commonly-used in vivo assays for angiogenesis are the rabbit corneal pocket, the hamster cheek pouch, and the chicken chorioallantoic membrane (CAM) assays. In each system an angiogenic substance is implanted in the cornea, cheek pouch, or the CAM, respectively, in order to induce angiogenesis. The corneal pocket and cheek pouch in vivo assays do not provide a clear view of the capillary system, including the endothelial cell walls. The CAM chick assay involves a species (the chicken) that is too distant to be useful for assessing angiogenic modulating agents and their effects in mammals including humans.
The pupillary membrane (PM) is a unique in vivo model system that has been discovered to have unique advantages as an in vivo assay for angiogenic modulators. This structure is a temporary vascular network that surrounds the anterior part of the lens in the developing eye. In humans, the PM is present only during embryogenesis as it regresses during the third trimester, although there are rare cases of a pupillary membrane persisting after birth (persistent pupillary membrane). In many species of the rodent family, regression occurs in the second week after birth. Being situated in the anterior chamber of the eye, the PM can be visualized vitally and is accessible for manipulation in vivo via trans-corneal injection. Since the PM is composed of a two-dimensional array of microcapillaries that can be rapidly dissected from the eye, this structure is uniquely suited to test the immediate in vivo response of microvessels to angiogenic modulators.
Therefore it is an object herein to provide an improved in vivo method for identifying compounds that modulate vascular and endothelial cell proliferation and inhibition. In particular, it is an object herein to provide an in vivo method for screening for both pro- and anti-modulators of angiogenesis. It is also an object to define a method for evaluating in vivo the angiogenic effect of small molecules and chemical substances.